The diagnosis of Alzheimer's disease, especially in the early stages, is notoriously difficult. Postmortem analysis of the brain shows the presence of neuritic plaques and neurofibulary tangles. Studies of brain tissue in the past have focused on gray matter pathology, but white matter has also been implicated in more recent studies. White matter in Alzheimer's subjects shows a dramatic loss of myelin and axons, DNA fragmentation, and plasmalogen deficiency in early stages of the disease. (“Plasmalogen” refers to a class of phospholipid enriched in myelin.) Most Alzheimer's patients show evidence of white matter degeneration and loss of oligodendrocytes (Morris, J. C., Neurology (1993) 43:2412–2414).
Sulfatides are a class of sulfated galactocerebrosides. A general structure for sulfatides is shown in FIG. 1. The various sulfatides differ only in the fatty acid represented by RCO in the structure. Sulfatides are synthesized through the mediation of ceramidase galactosyl transferase with subsequent sulfation mediated by galactocerebroside sulfotransferase. They are degraded, ultimately to ceramides, by the action of sulfatidase and galactosyl ceramidase. Sulfatides are important in the regulation of cell growth, protein trafficking, signal transduction, cell adhesion, neuronal plasticity and cell morphogenesis and are synthesized exclusively by oligodendrocytes in the central nervous system. They are present predominantly in the myelin sheath surrounding the axons and are present both in gray matter and white matter in the brain.
It has previously been shown that a deficiency in sulfatidase leads to an accumulation of sulfatides and to metachromatic leukodystrophy (Hess, B., et al., Proc. Natl. Acad. Sci. USA (1996) 93:14821–14826). A galactosyl ceramidase deficiency also leads to the accumulation of sulfatides as well as galactocebrosides in Krabbe's disease (Svennerholm, L., J. Lipid Res. (1980) 21:53–64). Further, knockout mice lacking ceramide galactosyl transferase show deficiencies in sulfatides and galactocerebrosides and generally die by three months and demonstrate abnormal axonal function. (Bosio, A., et al., Proc. Natl. Acad. Sci. USA (1996) 93:13280–13285; Coetzee, T., et al., Cell (1996) 86:209–219.)
It is also known that the ceramides, which are sulfatide degradation products are lipid second messengers and mediate inflammatory cytokines and growth factors so that the accumulation of ceramides results in up-regulation of cytokines, generation of reactive oxygen species, interruption of the mitochondrial respiratory chain and apoptosis (Hannun, Y. A., et al., Science (1996) 274:1855–1859; Kolesnick, R. N., et al., Ann. Rev. Physiol. (1998) 60:643–665; Paola, M. D., et al, Biochemistry (2000) 39:6660–6668).
Gottfries, C-G., et al., Int. Psycho-Geriatrics (1996) 8:365–372 describe studies which purport to distinguish the membrane components in brain of subjects with Alzheimer's disease from those with senile dementia of the Alzheimer's type (SDAT). SDAT and Alzheimer's disease are presently classified together since they appear to differ only in the age of onset of dementia; the onset in “pure Alzheimer's disease” is at an earlier age than that of SDAT. Nevertheless, this study showed that phospholipids, cholesterol, cerebroside and sulfatides were reduced in the frontal lobe white matter in the SDAT group as compared to age-matched controls and Alzheimer's patients. Levels of sulfatides in body fluids such as cerebrospinal fluid, blood, or urine in Alzheimer's patients or controls were not measured.
In a more recent publication, Han, X., et al., J. Neurochem. (2002) 82:809–818 further examined the status of sulfatide both in gray matter and white matter. This study, by the group of which the present inventors are members, showed that sulfatides were depleted both in gray matter and white matter in Alzheimer's subjects with very mild dementia, mild, and severe dementia, whereas other major classes of lipid, except plasmalogen were not altered. It was also established that there was no deficiency in the biosynthesis of sulfatides in these individuals and that the content of ceramides was elevated in the white matter. The authors concluded that the decrease in sulfatides was associated with Alzheimer's pathology even in very early stages in the gray and white matter of the brain. No measurements were taken of body fluids of any kind.
Fredman, P., et al., Acta Neurol. Scand. (1992) 85:103–106 measured sulfatides in the cerebrospinal fluid of patients with vascular dementia (i.e., dementia caused by stroke) and showed that the sulfatide concentrations in the patients with vascular dementia were at a significantly higher level than those in controls and in the Alzheimer's disease group. The levels in Alzheimer's patients and in controls appeared to be similar. The sulfatide levels in the group with vascular dementia were 307±118 nm/l while that in controls was 145±86 nm/l and in Alzheimer's patients 178±79 nm/l. The authors suggest that the elevation in sulfatide concentration reflects demyelination.
Alternative diagnoses for Alzheimer's disease and early stages thereof which measure components of biological fluids have been described in PCT publication WO 01/49875 and U.S. publication No. 2001/0044126A1 incorporated herein by reference. These documents describe assays in cerebrospinal fluid and blood which are predictive of Alzheimer's disease. These assays involve the levels of circulating amyloid-beta (Aβ) peptide in the blood and in the cerebrospinal fluid (CSF). The Aβ peptide in circulating form is composed of 39–43 amino acids (mostly 40 or 42 amino acids) and results from the cleavage of a common precursor, amyloid precursor protein, designated APP. It was found that the ratio of Aβ40 to Aβ42 is elevated in individuals with Alzheimer's disease or a propensity therefor. The total circulating levels of Aβ peptide, however, do not differ in Alzheimer's patients and in normal individuals. While statistically this method is helpful, there is some overlap in the values of this ratio between normal and Alzheimer's subjects.
It has also been shown that Aβ42 decreases and tau protein increases in patients with moderate to severe clinical Alzheimer's disease (Galasko, D., et al., Arc. Neurol. (1998) 55:937–945).
A potential dramatic improvement in this assay is described in U.S. Provisional Applications 60/313,221 filed 17 Aug. 2001 and 60/334,987 filed 23 Oct. 2001, and incorporated herein by reference. In this method, antibodies that sequester Aβ peptide from its bound circulating form in blood alter the clearance of soluble and bound forms of Aβ in the central nervous system and alter the levels of circulating Aβ peptides in the blood when the subject is in clinical or preclinical stage of Alzheimer's. Thus, the level of Aβ40, Aβ42 or the ratio of Aβ40/Aβ42 in the blood of the subject can be measured after injecting these antibodies and an elevation in any of these is indicative of Alzheimer's disease. This method, however, has not yet been assessed in human patients.
The present invention offers a method which clearly separates patients in even the very early stages of Alzheimer's disease from normal subjects. Virtually no overlap occurs between values obtained in subjects who are normal as compared to those with early stage Alzheimer's disease.